P434: MITOCONDRIAL MCL1 REGULATES LEUKEMIC CELLS METABOLISM VIA DIRECT INTERACTION WITH HEXOKINASE II. METABOLIC SIGNATURE AT ONSET PREDICTS OVERALL SURVIVAL IN AMLS’ PATIENTS
Authors: N. I. Noguera, G. Catalano, A. Zaza, C. Banella, T. Ottone, E. Pelosi, S. Travaglini, M. Divona, M. I. Del Principe, F. Buccisano, L. Maurillo, E. Amatuna, U. Testa, A. Venditti, M. T. Voso
Journal: HemaSphere (2022)

Background: Metabolic reprogramming is a basic feature in cancer. Leukemia associated cellular reprogramming, metabolic heterogeneity at onset and metabolic clonal evolution, driven by therapy, are essential insights that still remain unclear Aims: We compared Acute Myeloid Leukemias’ (AMLs) cells metabolism to hematopoietic progenitors’’ and normal maturing bone marrow cells’, to acquire useful prognostic information and to uncover actionable therapeutic targets. Methods: Methods We analyzed fresh primary blast from 19 AML patients, hematopoietic progenitors obtained from normal CD34+ cells differentiated to promyelocytes and granulocyte and the MV4-11, OCI-AML2 and OCI-AML3 cell lines, using a Seahorse Bioscience XFe96 analyzer. We silence MCL1 by siRNA and evaluated the interaction between HK2, MCL-1 and VDAC by co-immunoprecipitation and confocal microscopy. We assessed expression of MCL-1 and HK2 by western blot; HK2 by q-RTPCR. Results: Primary AML blast cells feature a lower spare respiratory capacity (SRC) (p=0.02) and lower glycolytic capacity (p=0.02) as compared to early progenitors/precursors (EP/P) from cultured CB CD34+ cells at day 7 of culture (N7, mostly promyelocytes) (Figure 1 a and b). Primary AML blast depend principally on fatty acids (p<0.05); they display a great flexibility, switching to glucose or glutamine to meet their energetic needs (Figure 1c). Consistent with the high adaptability of AML cells, and the emergence of resistance to therapy. We could define two populations (cut off value 10 pmol/min/x105 cells): one with higher (22±12 pmol/min/x105 cells) and on with lower (3±2 pmol/min/x105 cells) p<0.0001 levels of proton leak. The cases with higher proton leaks levels presented a reduced, extremely short, overall survival (p=0.048). We defined two SRC populations (cut off value 80 pmol/min/x105 cells): higher (124±47 pmol/min/x105 cells) and lower (52±25 pmol/min/x105cells) levels (p=0.0001). Interestingly the cases with higher SRC showed a trend of reduced overall survival. Associating high proton leak levels with high SRC the significance increases to p= 0.007. Considering the Basal OXPHOS of the AML patient’s cells, we observed two populations higher (54±12 pmol/min/x105 cells) and lower (16±6 pmol/min/x105cells) levels (p=0.0001). In patients with both high SRC plus high basal OXPHOS, the overall survival shortage is significant (p=0.002), indicating that an high SRC associated to higher basal respiration in AML blast cells confers greater aggressiveness and resistance to the therapy (Figure 1d). Patients with high mitochondrial respiration had a significantly higher myeloid cell leukemia 1 protein (MCL1) expression. We ascertained that MCL1 directly binds to Hexokinase II (HK2) on the outer mitochondrial membrane (OMM) affecting its stability. Image:Summary/Conclusion: We demonstrate that high proton leak and high mitochondrial respiration at onset, arguably with the concourse of MCL1/HK2 action, is significantly linked with a shorter overall survival in AMLs’ patients. Our data describe a new function of MCL1 protein in AMLs’ cells, forming a complex with HK2 co-localized to the voltage dependent anion channel (VDAC) on the OMM, thus promoting glycolysis and OXPHOS, ultimately conferring metabolic plasticity and promoting resistance to therapy. The lower spare respiratory capacity and lower glycolytic capacity of AML patients’ blast respect to normal early hematopoietic precursors suggest a therapeutic window to use glycolytic and mitochondrial inhibitors in resistant AML patients.